Congenital Hypogonadotropic Hypogonadism Due to GNRH Receptor Mutations in Three Brothers Reveal Sites Affecting Conformation and Coupling

Congenital hypogonadotropic hypogonadism (CHH) is characterized by low gonadotropins and failure to progress normally through puberty. Mutations in the gene encoding the GnRH receptor (GNRHR1) result in CHH when present as compound heterozygous or homozygous inactivating mutations. This study identifies and characterizes the properties of two novel GNRHR1 mutations in a family in which three brothers display normosmic CHH while their sister was unaffected. Molecular analysis in the proband and the affected brothers revealed two novel non-synonymous missense GNRHR1 mutations, present in a compound heterozygous state, whereas their unaffected parents possessed only one inactivating mutation, demonstrating the autosomal recessive transmission in this kindred and excluding X-linked inheritance equivocally suggested by the initial pedigree analysis. The first mutation at c.845 C>G introduces an Arg substitution for the conserved Pro 282 in transmembrane domain (TMD) 6. The Pro282Arg mutant is unable to bind radiolabeled GnRH analogue. As this conserved residue is important in receptor conformation, it is likely that the mutation perturbs the binding pocket and affects trafficking to the cell surface. The second mutation at c.968 A>G introduces a Cys substitution for Tyr 323 in the functionally crucial N/DPxxY motif in TMD 7. The Tyr323Cys mutant has an increased GnRH binding affinity but reduced receptor expression at the plasma membrane and impaired G protein-coupling. Inositol phosphate accumulation assays demonstrated absent and impaired Gαq/11 signal transduction by Pro282Arg and Tyr323Cys mutants, respectively. Pretreatment with the membrane permeant GnRHR antagonist NBI-42902, which rescues cell surface expression of many GNRHR1 mutants, significantly increased the levels of radioligand binding and intracellular signaling of the Tyr323Cys mutant but not Pro282Arg. Immunocytochemistry confirmed that both mutants are present on the cell membrane albeit at low levels. Together these molecular deficiencies of the two novel GNRHR1 mutations lead to the CHH phenotype when present as a compound heterozygote

Effects of dietary organic chromium on gilthead seabream (Sparus aurata L.) performances and liver microsomal metabolism

The effect of yeast and chromium yeast on gilthead seabream (Sparus auratus L) performance, carcass indices and body composition was studied. Whether supplementation affected liver microsomal mixed function oxidases using either multibioprobes (testosterone) or highly specific substrates to cytochrome P450 (CYP) isoforms was also investigated. Seabream juveniles (35-37 g initial weight) were allocated into 12 800 L tanks of 50 fish each for 8 7 days and fed pelleted experimental diets, i.e. control, yeast supplemented (1.6%) and chromium yeast supplemented at both low (800 p.p.b.) and high chromium level (53 810 p.p.b.). At the end of the experiment, growth, feed conversion ratio, thermal-unit growth coefficient, carcass yield, hepatosomatic index, and carcass and fillet proximate compositions were similar among treatments and only condition factor was statistically different. Organic chromium at both doses affected CYP-catalysed drug reactions slightly, as shown by the modest effect on the regio- and stereoselective hydroxylations of testosterone, as well as the metabolism of the selected probes. Overall, we found that chromium yeast did not change performance substantially, nor carcass indices, carcass and fillet chemical compositions, or hepatic xenobiotic metabolizing enzymes of gilthead seabream. © 2001 Blackwell Science Ltd

Two Novel Missense mutations in the GnRHR Gene in Two Siblings with Isolated Hypogonadotropic Hypogonadism

Introduction: Gonadotropin releasing hormone receptor (GnRHR) is a seven transmembrane G protein-copuled receptor (GPCR) that binds GnRH, the principal regulator of the reproductive function. Mutations in the GnRHR gene are associated with the autosomic recessive form of isolated hypogonadotropic hypogonadism (HH) characterized by variable forms of absent or incomplete pubertal development and infertility. The human GnRHR gene is located on chromosome 4q13.2-13.3. Its coding sequence comprises 3 exons and encodes a 328 amino acid protein with some particular features that distinguish it from other GPCRs. Objective: We report GnRHR molecular analysis in two Italian siblings presenting clinical signs of HH. Patients and Methods: The female was referred at age 14 for pubertal delay (no sexual secondary signs). The male was followed since prepuberty. He started pubertal development at age 13 and the testis reached a volume of 8 ml with no subsequent progression. Hormonal evaluation showed very low levels of both gonadotropins (LHRH test) and sexual steroids in both patients. GnRHR gene was amplified in three different fragments with primers complementary to flanking intronic sequences and directly sequenced by CEQ2000 (Beckman-Coulter) on both strands. Results: Direct sequencing of exon 1 revealed in the two siblings the presence of two new single nucleotide substitutions resulting in the Thr104Ile and the Tyr108Cys substitution in the first extracellular loop (ECL1), confirmed on both strands in two different PCR reactions. The molecular analysis confirmed the carrier status of the parents. Conclusions: We identified two new missense mutations in the GnRHR gene in two siblings with HH. The nature of substitutions (polar Thr with hydrophobic Ile, and aromatic Tyr with a Cys) in the ECL1 involved in the ligand-receptor interaction, as well as the high conservation of the two residues in all mammalian GnRHR, are suggestive of some implications in the phenotype observed

Identification of rare alleles in an Italian population of 284 patients with 21-hydoxylase deficiency by complete sequencing of the CYP21 gene.

In the paper the authors described the identification of rare alleles in an Italian population of 284 patients with 21-hydoxylase deficiency by complete sequencing of the CYP21 gene

Hydroxyl radical generation, levels of tumor necrosis factor-alpha, and progression to heart failure after acute myocardial infarction

OBJECTIVES We used acetylsalicylic acid (ASA) as a probing agent to quantify hydroxyl radical (˙OH) in Controls and patients with coronary artery disease and to prospectively investigate ˙OH production in patients with myocardial infarction (MI) complicated by heart failure (HF). BACKGROUND Oxidative stress status (OSS) is a mechanism for transition to HF in experimental heart injury models, but evidence for its causal role in humans is still limited. METHODS Thirty healthy subjects (Controls), 12 patients with stable angina (Group 1), and 74 patients with ST-segment elevation MI (Group 2) were enrolled. A dose of 250 mg Flectadol was given intravenously before each blood collection to determine the 2,3-dihydroxybenzoic acid/salicylic acid (DHBA/SA) ratio. We also quantified vitamin E and coenzyme Q10 to monitor antioxidant reserve, as well as tumor necrosis factor (TNF)-alpha, TNF-soluble receptors, interleukin (IL)-6, and IL-1ra to assess inflammatory status. All measurements were repeated at month 6 in Group 2. RESULTS There were no differences between Controls and Group 1. Group 2 showed increased ˙OH production, peaking at 24 h, whereas vitamin E and coenzyme Q10 progressively declined. Group 2 patients developing HF during hospitalization (Group 2Bi) presented with an increase of both ˙OH production at discharge and inflammatory status, as compared with patients without HF (Group 2Ai), persisting at month 6 in post-MI patients with HF (Group 2Bii). CONCLUSIONS We found a distinct pattern of ˙OH generation in post-MI patients who show progression to HF. The interplay between OSS and inflammatory status should be targeted as a possible mechanism of progression to post-MI left ventricular dysfunction. (J Am Coll Cardiol 2004;43:2000–8) © 2004 by the American College of Cardiology Foundatio